1. Field of the Invention
The present invention relates to the use of compounds acting as inhibitors of enzymes having histone deacetylase activity for the medical therapy of conditions which predispose a person for the development of a disease, such as but not limited to cancer, inflammatory or metabolic diseases. Such conditions are linked to genetically inherited mutations of crucial genes which predispose a person with this condition to develop the disease phenotype. Thus, the invention relates to the use of such compounds for a suppressive therapeutic approach—the SUPPRESSION THERAPY—in order to inhibit or delay the onset or progression of the genetically predisposed disorder. Furthermore, the invention includes the manufacture of a clinically used medicament for the SUPPRESSION THERAPY of such inherited predisposing conditions.
Current progress in the understanding of the molecular biology of pathogenic processes such as modern tumor biology has provided insights into the genetic basis and into the fundamental biochemical pathways of the development of many diseases, e.g., of cancerogenesis. These newly identified mechanisms offer new opportunities for therapeutic intervention not only in the management of the acute disease state, but also for the medical management of a pre-symptomatic condition using an approach which herein is referred to as Suppression Therapy. Such a condition is defined by the inherited presence of gene mutations or critical gene polymorphisms which predispose a person to develop the disease phenotype. Suppression therapy is a novel concept, which refers to the inhibition or delay of pathogenesis through the use of naturally occurring or synthetic compounds and drugs which display such suppressive properties, e.g., by suppression of the mechanisms caused by such inherited genetic mutations, and thus, suppression of diseased signal transductions and the development of the disease phenotype.
In this invention, we propose the use of inhibitors of enzymes having histone deacetylase activity in medical suppression therapy of a set of human conditions, which are based on inherited mutations that predispose a person to develop a disorder and in which the development of the disease is linked to the presence of such mutations or polymorphisms.
2. Brief Description of the Related Art
Chromatin Regulation and Disease
Local remodeling of chromatin is a key step in the transcriptional activation of genes. Dynamic changes in the nucleosomal packaging of DNA must occur to allow transcriptional proteins to make contact with the DNA template. One of the most important mechanisms influencing chromatin remodeling and gene transcription are posttranslational modifications of histones and other cellular proteins by acetylation and subsequent changes in chromatin structure (Davie, 1998, Curr Opin Genet Dev 8, 173-8; Kouzarides, 1999, Curr Opin Genet Dev 9, 40-8; Strahl and Allis, 2000, Nature 403, 41-4). In the case of histone hyperacetylation, changes in the electrostatic attraction of DNA and steric hindrance introduced by the hydrophobic acetyl group leads to destabilisation of the interaction of histones with DNA. As a result, acetylation of histones disrupts nucleosomes and allows the DNA to become accessible to the transcriptional machinery. Removal of the acetyl groups allows the histones to bind more tightly to DNA and to adjacent nucleosomes, and thus, to maintain a transcriptionally repressed chromatin structure. Acetylation is mediated by a series of enzymes with histone acetyltransferase (HAT) activity. Conversely, acetyl groups are removed by specific histone deacetylase (HDAC) enzymes. Disruption of these mechanisms gives rise to transcriptionally misguided regulation and may contribute to a variety of human diseases, including autoimmune, inflammatory, metabolic or hyperproliferative disorders, including tumorigenic transformation and tumor progression.
Additionally, other molecules such as transcription factors alter their activity and stability depending on their acetylation status. E.g. PML-RAR, the fusion protein associated with acute promyelocytic leukemia (APL) inhibits p53 through mediating deacetylation and degradation of p53, thus allowing APL blasts to evade p53 dependent cancer surveillance pathways. Expression of PML-RAR in hematopoietic precursor cells results in repression of p53 mediated transcriptional activation and protection from p53-dependent apoptosis triggered by genotoxic stresses (X-rays, oxidative stress). However, the function of p53 is reinstalled in the presence of HDAC inhibitors implicating active recruitment of HDAC to p53 by PML-RAR as the mechanism underlying p53 inhibition (Insinga et al., February 2004, EMBO Journal, 1-11). Therefore, acetylation of proteins distinct from histones, such as acetylation of p53, plays a crucial role in the anti-disease activity of HDAC inhibitors.
Nuclear Receptors and Histone Deacetylases
Nuclear hormone receptors are ligand-dependent transcription factors that control development and homeostasis through both positive and negative control of gene expression. Defects in these regulatory processes underlie the causes of many diseases and play an important role in the development of cancer. Many nuclear receptors, including T3R, RAR and PPAR, can interact with corepressors, such as N-CoR and SMRT, in the absence of a ligand and thereby inhibit transcription. Furthermore, N-CoR has also been reported to interact with antagonist-occupied progesterone and estrogen receptors. Most interestingly, N-CoR and SMRT have been shown to exist in large protein complexes, which also contain mSin3 proteins and histone deacetylases (Pazin and Kadonaga, 1997; Cell 89, 325-8). Thus, the ligand-induced switch of nuclear receptors from repression to activation reflects the exchange of corepressor and coactivator complexes with antagonistic enzymatic activities.
Gene Regulation by Nuclear Receptors
Such corepressor complexes which contain HDAC activity, not only mediate repression by nuclear receptors, but also interact with additional transcription factors including Mad-1, BCL-6, and ETO. Many of these proteins play key roles in disorders of cell proliferation and differentiation (Pazin and Kadonaga, 1997, Cell 89, 325-8; Huynh and Bardwell, 1998, Oncogene 17, 2473-84; Wang, J. et al., 1998, Proc Natl Acad Sci U S A 95, 10860-5). T3R for example was originally identified on the basis of its homology with the viral oncogene v-erbA, which in contrast to the wild type receptor does not bind a ligand and functions as a constitutive repressor of transcription. Furthermore, mutations in RARs have been associated with a number of human cancers, particularly acute promyelocytic leukemia (APL) and hepatocellular carcinoma. In APL patients RAR fusion proteins resulting from chromosomal translocations involve either the promyelocytic leukemia protein (PML) or the promyelocytic zinc finger protein (PLZF). Although both fusion proteins can interact with components of the corepressor complex, the addition of retinoic acid dismisses the corepressor complex from PML-RAR, whereas PLZF-RAR interacts constitutively. These findings provide an explanation why PML-RAR APL patients achieve complete remission following retinoic acid treatment whereas PLZF-RAR APL patients respond very poorly (Grignani et al., 1998, Nature 391, 815-8; Guidez et al., 1998, Blood 91, 2634-42; He et al., 1998, Nat Genet 18, 126-35; Lin et al., 1998, Nature 391, 811-4).
Recently, a PML-RAR patient who had experienced multiple relapses after treatment with retinoic acid has been treated with the HDAC inhibitor phenylbutyrate, resulting in complete remission of the leukemia (Warrell et al., 1998, J. Natl. Cancer Inst. 90, 1621-1625).
The Protein Family of Histone Deacetylases
The recruitment of histone acetyltranferases (HATs) and histone deacetylases (HDACs) is considered as a key element in the dynamic regulation of many genes playing important roles in cellular proliferation and differentiation. Hyperacetylation of the N-terminal tails of histones H3 and H4 correlates with gene activation whereas deacetylation can mediate transcriptional repression. Consequently, many diseases have been linked to changes in gene expression caused by mutations affecting transcription factors. Aberrant repression by leukemia fusion proteins such as PML-RAR, PLZF-RAR, AML-ETO, and Stat5-RAR serves as a prototypical example in this regard. In all of these cases, chromosomal translocations convert transcriptional activators into repressors, which constitutively repress target genes important, e.g., for hematopoietic differentiation via recruitment of HDACs. It is plausible that similar events could also contribute to pathogenesis in many other types of diseases. There is growing evidence that the same holds true also for autoimmune, inflammatory, metabolic or hyperproliferative disorders.
Mammalian histone deacetylases can be divided into three subclasses (Gray and Ekström, 2001). HDACs 1, 2, 3, and 8 which are homologues of the yeast RPD3 protein constitute class I. HDACs 4, 5, 6, 7, 9, and 10 are related to the yeast Hda 1 protein and form class II. Recently, several mammalian homologues of the yeast Sir2 protein have been identified forming a third class of deacetylases which are NAD dependent. Furthermore, HDAC 11 has been classified as a class I histone deacetylase with structural features of a class II HDAC. All of these HDACs appear to exist in the cell as subunits of a plethora of multiprotein complexes. In particular, class I and II HDACs have been shown to interact with transcriptional corepressors mSin3, N-CoR and SMRT which serve as bridging factors required for the recruitment of HDACs to transcription factors.
Therapy with HDAC Inhibitors
Additional clinical investigations have recently been initiated to exploit the systemic clinical treatment of cancer patients based on the principle of HDAC inhibition. By now, a clinical phase II trial with the closely related butyric acid derivative Pivanex (Titan Pharmaceuticals) as a monotherapy has been completed demonstrating activity in stage III/IV non-small cell lung cancer (Keer et al., 2002, ASCO, Abstract No. 1253). More HDAC inhibitors have been identified, with NVP-LAQ824 (Novartis) and SAHA (Aton Pharma Inc.) being members of the structural class of hydroxamic acids tested in phase II clinical trials (Marks et al., 2001, Nature Reviews Cancer 1, 194-202). Another class comprises cyclic tetrapeptides, such as depsipeptide (FR901228—Fujisawa) used successfully in a phase II trial for the treatment of T-cell lymphomas (Piekarz et al., 2001, Blood 98, 2865-8). Furthermore, MS-27-275 (Mitsui Pharmaceuticals), a compound related to the class of benzamides, is now being tested in a phase I trial treating patients with hematological malignancies.
2-propyl-pentanoic acid
2-propyl-pentanoic acid (2PPA) has multiple biological activities which depend on different molecular mechanisms of action:                2PPA is an antiepileptic drug.        2PPA is teratogenic. When used as an antiepileptic drug during pregnancy, 2PPA can induce birth defects (neural tube closure defects and other malformations) in a few percent of born children. In mice, 2PPA is teratogenic in the majority of mouse embryos when properly dosed.        2PPA activates a nuclear hormone receptor (PPARδ). Several additional transcription factors are also derepressed, but some factors are not significantly derepressed (glucocorticoid receptor, PPARα).        
2PPA occasionally causes hepatotoxicity, which may depend on poorly metabolized esters with coenzyme A.
2PPA is an inhibitor of HDACs.
The use of 2PPA derivatives allowed to determine that the different activities are mediated by different molecular mechanisms of action. Teratogenicity and antiepileptic activity follow different modes of action because compounds could be isolated which act either preferentially teratogenic or preferentially antiepileptic (Nau et al., 1991, Pharmacol. Toxicol. 69, 310-321). Activation of PPARδ was found to be strictly correlated with teratogenicity (Lampen et al., 1999, Toxicol. Appl. Pharmacol. 160, 238-249) suggesting that, both, PPARδ activation and teratogenicity require the same molecular activity of 2PPA. Also, differentiation of F9 cells strictly correlated with PPARδ activation and teratogenicity as suggested by Lampen et al., 1999, and documented by the analysis of differentiation markers (Werling et al., 2001, Mol. Pharmacol. 59, 1269-1276). It was shown, that PPARδ activation is caused by the HDAC inhibitory activity of 2PPA and its derivatives (WO 02/07722 A2; WO 03/024442 A2). Furthermore, it was shown that the established HDAC inhibitor TSA activates PPARδ and induces the same type of F9 cell differentiation as 2PPA. From these results it can be concluded that not only activation of PPARδ but also induction of F9 cell differentiation and teratogenicity of 2PPA or 2PPA derivatives are caused by HDAC inhibition.
Antiepileptic and sedating activities follow different structure activity relationships and thus obviously depend on a primary 2PPA activity distinct from HDAC inhibition. The mechanism of hepatotoxicity is poorly understood and it is unknown whether it is associated with formation of the 2PPA-CoA ester. HDAC inhibition, however, appears not to require CoA ester formation.
2PPA as an Inhibitor of Histone Deacetylases
2PPA has been developed as a drug used for the treatment of epilepsia. Accordingly, 2PPA is used systemically, orally, or intravenously, to allow the drug to pass the blood brain barrier to reach the epileptic target regions in the brain tissue in order to fulfill its anti-epileptic mission. Moreover, 2PPA has been shown to potentially possess particular beneficial effects when used for the treatment of many different types of human cancers as a single agent or in combination with a whole variety of other anti-tumor therapies which are individually based on strikingly different modes of action, by inhibiting specific sets of enzymes having HDAC activity and thereby inducing differentiation and/or apoptosis (WO 02/07722 A2, EP 1170008; WO 03/024442 A2, EP 1293205 A1). For the treatment or prevention of malignant diseases, autoimmune diseases, or other inflammatory or hyperproliferative disorders, 2PPA may also be administered systemically, orally, or intravenously. Furthermore, it was shown, that 2PPA permeates human skin effectively and therefore can be administered topically on skin exhibiting beneficial effects when used for the topical treatment or prevention of autoimmune, inflammatory or hyperproliferative human skin diseases, e.g., psoriasis and human skin cancer (EP application No. 03014278.0). This new potential of 2PPA, and other HDAC inhibitors, to act as immune modulating compounds supports this invention to employ these compounds as anti-inflammatory drugs for the therapy of disorders linked to pathologically overactive immune cells.